Electron Transfer Binding Site of Cytochrome c to Carboxylic Acid-Terminated Alkanethiol Self-assembled Monolayers Immobilized on Gold Electrode

It is proposed that Lys-13 of mammalian cytochrome cfacilitates the most efficient electron transfer (ET) pathway to the carboxylate terminus of alkanethiol self-assembled monolayers (SAM) on gold electrodes. In order to confirm the proposed ET pathway, the ET reaction rate of a rat cytochrome c mutant (RC9K13A), in which lysine-13 is replaced by alanine, is measured at a 3-mercaptopropionate SAM on a gold electrode. The ET rate of K13A is more than six orders of magnitude smaller than that of the native one. In the mutant, Ala-13 can no longer facilitate the ET pathway. Based on the measurements, the potential candidate for the binding site of RC9K13A is Lys-8.     

Mimicking protein-protein electron transfer: voltammetry of Pseudomonas aeruginosa azurin and the Thermus thermophilus Cu(A) domain at omega-derivatized self-assembled-monolayer gold electrodes

Well-defined voltammetric responses of redox proteins with acidic-to-neutral pI values have been obtained on pure alkanethiol as well as on mixed self-assembled-monolayer (SAM) omega-derivatized alkanethiol/gold bead electrodes. Both azurin (P. aeruginosa) (pI = 5.6) and subunit II (Cu(A) domain) of ba(3)-type cytochrome c oxidase (T. thermophilus) (pI = 6.0) exhibit optimal voltammetric responses on 1:1 mixtures of [H(3)C(CH(2))(n)()SH + HO(CH(2))(n)()SH] SAMs. The electron transfer (ET) rate vs distance behavior of azurin and Cu(A) is independent of the omega-derivatized alkanethiol SAM headgroups. Strikingly, only wild-type azurin and mutants containing Trp48 give voltammetric responses: based on modeling, we suggest that electronic coupling with the SAM headgroup (H(3)C- and/or HO-) occurs at the Asn47 side chain carbonyl oxygen and that an Asn47-Cys112 hydrogen bond promotes intramolecular ET to the copper. Inspection of models also indicates that the Cu(A) domain of ba(3)-type cytochrome c oxidase is coupled to the SAM headgroup (H(3)C- and/or HO-) near the main chain carbonyl oxygen of Cys153 and that Phe88 (analogous to Trp143 in subunit II of cytochrome c oxidase from R. sphaeroides) is not involved in the dominant tunneling pathway. Our work suggests that hydrogen bonds from hydroxyl or other proton-donor groups to carbonyl oxygens potentially can facilitate intermolecular ET between physiological redox partners.     

Determination of self-exchange rate of alkanethiolates in self-assembled monolayers on gold using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

In this paper, we describe a new method for determining the exchange rates of alkanethiolates in self-assembled monolayers (SAMs) on gold using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze the compositions of the alkanethiolate in SAMs rapidly and directly. In particular, to investigate the self-exchange of alkanethiols, we prepared a deuterated alkanethiol that has the same molecular properties as the non-deuterated alkanethiol but a different molecular weight. SAMs consisting of deuterated alkanethiolates were immersed in a solution of the non-deuterated alkanethiol, and the influences of the immersion time, temperature, concentration, and solvent on the self-exchange rates were investigated. Furthermore, we assessed the exchange rates among alkanethiols with different carbon chain lengths and different size of ethylene glycol units. In addition, we performed molecular dynamics simulations using a model SAM system in order to understand the molecular mechanism of the exchange process.     

Effects of changes in the interparticle separation induced by alkanethiols on the surface plasmon band and other properties of nanocrystalline gold films

Effects of changing the interparticle separation on the surface plasmon bands of ultrathin films of gold nanoparticles have been investigated by examining the interaction of alkanethiols of varying chain length on nanocrystalline gold films generated at the organic-aqueous interface. Adsorption of alkanethiols causes blue-shifts of the surface plasmon adsorption band, the magnitude of the shift being proportional to the chain length. The disordered nanocrystals thus created (lambdamax, 530 m) are in equilibrium with the ordered nanocrystals in the film (lambdamax, 700 m) as indicated by an isosbestic point around 600 nm. Long chain thiols disintegrate or disorder the gold films more effectively, as demonstrated by the increased population of the thiol-capped gold nanocrystals in solution. The rate of interaction of the thiols with the film decreases with the decreasing chain length. The effect of an alkanethiol on the spectrum of the gold film is specific, in that the effects with long and short chains are reversible. The changes in the plasmon band of gold due to interparticle separation can be satisfactorily modeled on the basis of the Maxwell-Garnett formalism. Spectroscopic studies, augmented by calorimetric measurements, suggest that the interaction of alkanethiols involves two steps, the first step being the exothermic gold film-thiol interaction and the second step includes the endothermic disordering process followed by further thiol capping of isolated gold particles.     

Adsorption and thermal reactions of alkanethiols on pt(111): Dependence on the length of the alkyl chain

The adsorption and thermal decomposition of alkanethiols (R-SH, where R = CH3, C2H5, and C4H9) on Pt(111) were studied with temperature-programmed desorption (TPD) and X-ray photoelectron spectroscopy (XPS) with synchrotron radiation. Dissociation of sulfhydryl hydrogen (RS-H) of alkanethiol results in the formation of alkanethiolate; the extent of dissociation at an adsorption temperature of 110 K depends on the length of the alkyl chain. At small exposure, all chemisorbed CH3SH, C2H5SH, and C4H9SH decompose to desorb hydrogen below 370 K and yield carbon and sulfur on the surface. Desorption of products containing carbon is observed only at large exposure. In thermal decomposition, alkanethiolate is proposed to undergo a stepwise dehydrogenation: R'-CH2S --> R'-CHS --> R'-CS, R' = H, CH3, and C3H7. Further decomposition of the R'-CS intermediate results in desorption of H2 at 400-500 K and leaves carbon and sulfur on the surface. On the basis of TPD and XPS data, we conclude that the density of adsorption of alkanethiol decreases with increasing length of the alkyl chain. C4H9SH is proposed to adsorb mainly with a configuration in which its alkyl group interacts with the surface; this interaction diminishes the density of adsorption of alkanethiols but facilitates dehydrogenation of the alkyl group.     

Understanding the mechanism of short-range electron transfer using an immobilized cupredoxin

The hydrophobic patch of azurin (AZ) from Pseudomonas aeruginosa is an important recognition surface for electron transfer (ET) reactions. The influence of changing the size of this region, by mutating the C-terminal copper-binding loop, on the ET reactivity of AZ adsorbed on gold electrodes modified with alkanethiol self-assembled monolayers (SAMs) has been studied. The distance-dependence of ET kinetics measured by cyclic voltammetry using SAMs of variable chain length, demonstrates that the activation barrier for short-range ET is dominated by the dynamics of molecular rearrangements accompanying ET at the AZ-SAM interface. These include internal electric field-dependent low-amplitude protein motions and the reorganization of interfacial water molecules, but not protein reorientation. Interfacial molecular dynamics also control the kinetics of short-range ET for electrostatically and covalently immobilized cytochrome c. This mechanism therefore may be utilized for short-distance ET irrespective of the type of metal center, the surface electrostatic potential, and the nature of the protein-SAM interaction.     

Using scanning electrochemical microscopy (SECM) to measure the electron-transfer kinetics of cytochrome c immobilized on a COOH-terminated alkanethiol monolayer on a gold electrode

Cytochrome c was electrostatically immobilized onto a COOH-terminated alkanethiol self-assembled monolayer (SAM) on a gold electrode at ionic strengths of less than 40 mM. Scanning electrochemical microscopy (SECM) was used to simultaneously measure the electron transfer (ET) kinetics of the bimolecular ET between a solution-based redox mediator and the immobilized protein and the tunneling ET between the protein and the underlying gold electrode. Approach curves were recorded with ferrocyanide as a mediator at different coverages of cytochrome c and at different substrate potentials, allowing the measurement of k(BI) = 2 x 10(8) mol(-1) cm3 s(-1) for the bimolecular ET and k degrees = 15 s(-1) for the tunneling ET. The kinetics of ET was also found to depend on the immobilization conditions of cytochrome c: covalent attachment gave slightly slower tunneling ET values, and a mixed CH3/COOH-terminated ML gave faster tunneling ET rates. This is consistent with previous studies and is believed to be related to the degree of mobility of cyt c in its binding configuration and its orientation with respect to the underlying electrode surface.     

Scanning electrochemical microscopy. 59. Effect of defects and structure on electron transfer through self-assembled monolayers

Electron transfer (ET) rate kinetics through n-alkanethiol self-assembled monolayers (SAMs) of alkanethiols of different chain lengths [Me(CH2)nSH; n=8, 10, 11, 15] on Au and Hg surfaces and ferrocene (Fc)-terminated SAMs (poly-norbornylogous and HS(CH2)12CONHCH2Fc) on Au were studied using cyclic voltammetry and scanning electrochemical microscopy (SECM). The SECM results allow determination of the ET kinetics of solution-phase Ru(NH3)63+/2+ through the alkanethiol SAMs on Au and Hg. A model using the potential dependence of the measured rate constants is proposed to compensate for the pinhole contribution. Extrapolated values of koML for Ru(NH3)63+/2+ using the model follow the expected exponential decay (beta is 0.9) for different chain lengths. For a Fc-terminated poly-norbornyl SAM, the standard rate constant of direct tunneling (ko is 189+/-31 s(-1)) is in the same order as the ko value of HS(CH2)12CONHCH2Fc. In blocking and Fc SAMs, the rates of ET are demonstrated to follow Butler-Volmer kinetics with transfer coefficients alpha of 0.5. Lower values of alpha are treated as a result of the pinhole contribution. The normalized rates of ET are 3 orders of magnitude higher for Fc-terminated than for blocking monolayers. Scanning electron microscopy imaging of Pd nanoparticles electrochemically deposited in pinholes of blocking SAMs was used to confirm the presence of pinholes.     

Proton-coupled electron transfer of cytochrome c.

Cytochrome c (Cyt-c) was electrostatically bound to self-assembled monolayers (SAM) on an Ag electrode, which are formed by omega-carboxyl alkanethiols of different chain lengths (C(x)). The dynamics of the electron-transfer (ET) reaction of the adsorbed heme protein, initiated by a rapid potential jump to the redox potential, was monitored by time-resolved surface enhanced resonance Raman (SERR) spectroscopy. Under conditions of the present experiments, only the reduced and oxidized forms of the native protein state contribute to the SERR spectra. Thus, the data obtained from the spectra were described by a one-step relaxation process yielding the rate constants of the ET between the adsorbed Cyt-c and the electrode for a driving force of zero electronvolts. For C(16)- and C(11)-SAMs, the respective rate constants of 0.073 and 43 s(-1) correspond to an exponential distance dependence of the ET (beta = 1.28 A(-1)), very similar to that observed for long-range intramolecular ET of redox proteins. Upon further decreasing the chain length, the rate constant only slightly increases to 134 s(-1) at C(6)- and remains essentially unchanged at C(3)- and C(2)-SAMs. The onset of the nonexponential distance dependence is paralleled by a kinetic H/D effect that increases from 1.2 at C(6)- to 4.0 at C(2)-coatings, indicating a coupling of the redox reaction with proton-transfer (PT) steps. These PT processes are attributed to the rearrangement of the hydrogen-bonding network of the protein associated with the transition between the oxidized and reduced state of Cyt-c. Since this unusual kinetic behavior has not been observed for electron-transferring proteins in solution, it is concluded that at the Ag/SAM interface the energy barrier for the PT processes of the adsorbed Cyt-c is raised by the electric field. This effect increases upon reducing the distance to the electrode, until nuclear tunneling becomes the rate-limiting step of the redox process. The electric field dependence of the proton-coupled ET may represent a possible mechanism for controlling biological redox reactions via changes of the transmembrane potential.     

Open tubular capillary electrochromatography using capillaries coated with films of alkanethiol-self-assembled gold nanoparticle layers

We have used alkanethiol self-assembly and dithiol layer-by-layer (LBL) self-assembly processes to prepare an Au nanoparticle (NP)-coated open tubular capillary electrochromatography (OTCEC) column for the separation of three neutral steroid drugs (testosterone, progesterone, and testosterone propionate). The CEC column was fabricated through LBL self-assembly of Au NPs on a 3-aminopropyltrimethoxysilane (APTMS)-modified fused-silica capillary and subsequent surface functionalization of the Au NPs through self-assembly of alkanethiols. We investigated the electrochromatographic properties of the resulting Au NP-coated CEC column using a "reversed phase" test mixture of three steroid drugs. We found that the key factors affecting the separation performance were the number of Au NP layers, the length of the carbon-atom chain of the alkanethiol self-assembled on the Au NPs, the percentage of organic modifier, and the pH of the running electrolyte. This study reveals that the self-assembly of alkanethiols and dithiols onto Au NPs provides stationary phases for CEC separation that are easy to prepare and whose retention behavior is highly controllable and reproducible. We believe that our findings will contribute to further studies of the application of nanotechnology to separation science.     

12-Mercaptododecyl β-maltoside-modified gold nanoparticles: specific ligands for concanavalin A having long flexible hydrocarbon chains

A simple and highly specific protein detection system using glycoconjugated gold nanoparticles was investigated. This system was based on the aggregation of gold nanoparticles coated with carbohydrate alkanethiols in the presence of corresponding proteins (lectins) that had specific recognition for certain carbohydrates. In order to construct an efficient specific recognition system, maltoside alkanethiol was adopted as an effective sensing modifier having a disaccharide group and a flexible long alkyl chain. The surface modification of gold nanoparticles with maltoside alkanethiol resulted in a shift and broadening (from 520 to 610 nm) of the absorption peak. Monodispersed maltoside-adsorbed gold nanoparticles aggregated with the specific lectin, concanavalin A (Con A). This phenomenon was used to detect the presence of Con A and to estimate concentrations of Con A in sample solutions. The precipitate of the maltoside–gold nanoparticle–Con A mixture was redispersed by addition of methyl α-D-mannopyranoside whose adsorption coefficient is larger than that of maltoside with Con A.     

Studying electron transfer through alkanethiol self-assembled monolayers on a hanging mercury drop electrode using potentiometric measurements

A new approach based on measuring the change of the open-circuit potential (OCP) of a hanging mercury drop electrode (HMDE), modified with alkanethiols of different chain length conducted in a solution containing a mixture of Ru(NH3)6(2+) and Ru(NH3)6(3+) is used for studying electron transfer across the monolayer. Following the time dependence of the OCP allowed the extraction of the kinetic parameters, such as the charge transfer resistance (R(ct)) and the electron transfer rate constant (k(et)), for different alkanethiol monolayers. An electron tunneling coefficient, beta, of 0.9 A(-1) was calculated for the monolayers on Hg.     

Blocking behavior of self-assembled monolayers on gold electrodes

Self-assembled monolayers (SAMs) with metal electrodes, especially thiols on gold, are the subject of this investigation because of the unique properties of SAM-modified surfaces. Normal alkanethiols are used to modify the surface of a conventional gold electrode to block certain ions such as Pb(II) and Cu(II) from the surface of the electrode. Normal alkanethiols are also used to study the SAM-gold interfacial adsorption-desorption behavior of the self-assembled monolayer. The effects of varying chain length of SAMs, varying concentration of the alkanethiol solutions, immersion time of the pure gold electrode in the SAM solution, and the stability of a SAM-modified gold electrode in fresh chloroform are investigated using the oxidation-reduction peaks of gold. Conditions that optimize the surface coverage and the uniformity of the SAMs have been determined. Normal alkanethiols proved to be a good insulator on the electrode surface. Received: 16 January 1997 / Accepted: 4 March 1997     

Adsorption of ethoxylated cationic surfactants on self-assembled monolayers of alkanethiols on gold using surface plasmon resonance detection

Adsorption of a series of ethoxylated cationic surfactants at model surfaces of alkanethiol self-assembled monolayers was studied by the surface plasmon resonance technique. Model surfaces were tailor-made by choosing alkanethiols or mixtures of alkanethiols with methyl, hydroxyl, carboxyl, and trimethylammonium groups in terminal position. The ethoxylated and quaternized cationic surfactants having from 2 to 18 oxyethylene units, showed a decrease in adsorbed amount with increasing oxyethylene chain length for both hydrophobic and hydrophilic surfaces. On a negatively charged surface, containing carboxylate groups, the surfactant with only two oxyethylene groups adsorbed strongly due to electrostatic attraction and the adsorption increased with increasing amount of surface carboxylate groups. This work shows the usefulness of self-assembled alkanethiols on gold as a tool for performing surfactant adsorption studies on surfaces with variable hydrophobicity and charge.     

Dielectric properties and frequency response of self-assembled monolayers of alkanethiols

This paper presents dielectric properties of alkanethiol self-assembled monolayers (SAMs) under an ac electric field. Using a Hg-SAM/SAM-Hg junction, we measured the ac impedance of alkanethiol SAMs using a sinusoidal perturbation of 30 mV (peak-to-peak) with frequency ranging from 1 Hz to 1 MHz at zero bias. Semicircles at higher frequencies and at middle frequencies along with Warburg lines at lower frequencies were observed in complex plane impedance plots, that is, Nyquist plots. The frequency response of SAMs was analyzed by modeling the junction using an equivalent circuit and fitting the Nyquist plots. The semicircles at higher frequencies are attributed to the effect of the SAM/SAM interfaces, and the ones at middle frequencies are attributed to the effect of alkanethiol SAMs. The comparison in the plots of the imaginary part of the impedance Z against frequency for the bare Hg electrodes (in pure ethanal) and the SAM-covered Hg electrodes (in alkanethiol solution) supports the analysis. The Warburg lines are attributed to a certain ionic impurity. The dielectric loss spectra are further analyzed. Chain-length-dependent peaks, which correspond to different relaxation mechanisms, at higher frequencies and middle frequencies were observed in the spectra of the dissipation factor (tan delta vs frequency). The peaks move to small frequency with the increase of chain length of alkanethiols. Using a correlation of peak position with the chain length, we then derived active energies of 39-99 meV for alkanethiol SAMs of C7-C18 under an ac electric field.     

Large-scale molecular dynamics simulations of alkanethiol self-assembled monolayers

Large-scale molecular dynamics simulations of self-assembled alkanethiol monolayer systems have been carried out using an all-atom model involving a million atoms to investigate their structural properties as a function of temperature, lattice spacing, and molecular chain length. Our simulations show that the alkanethiol chains of 13-carbons tilt from the surface normal by a collective angle of 25 degrees along next-nearest-neighbor direction at 300 K. The tilt structure of 13-carbon alkanethiol system is found to depend strongly on temperature and exhibits hysteresis. At 350 K the 13-carbon alkanethiol system transforms to a disordered phase characterized by small collective tilt angle, flexible tilt direction, and random distribution of backbone planes. The tilt structure also depends on lattice spacing: With increasing lattice spacing a the tilt angle increases rapidly from a nearly zero value at a=4.7 A to as high as 34 degrees at a=5.3 A at 300 K for 13-carbon alkanethiol system. Finally, the effects of the molecular chain length on the tilt structure are significant at high temperatures.     

Direct electrochemistry of heme multicofactor-containing enzymes on alkanethiol-modified gold electrodes

Direct electrochemistry of heme multicofactor-containing enzymes, e.g., microbial theophylline oxidase (ThOx) and D-fructose dehydrogenase (FDH) from Gluconobacter industrius was studied on alkanethiol-modified gold electrodes and was compared with that of some previously studied complex heme enzymes, specifically, cellobiose dehydrogenase (CDH) and sulphite oxidase (SOx). The formal redox potentials for enzymes in direct electronic communication varied for ThOx from -112 to -101 mV (vs. Ag|AgCl), at pH 7.0, and for FDH from -158 to -89 mV, at pH 5.0 and pH 4.0, respectively, on differently charged alkanethiol layers. Direct and mediated by cytochrome c electrochemistry of FDH correlated with the existence of two active centres in the protein structure, i.e., the heme and the pyrroloquinoline quinone (PQQ) prosthetic groups. The effect of the alkanethiols of different polarity and charge on the surface properties of the gold electrodes necessary for adsorption and orientation of ThOx, FDH, CDH and SOx, favourable for the efficient electrode-enzyme electron transfer reaction, is discussed.     

Mixed self-assembled monolayers of terminally functionalized thiols at gold surfaces characterized by angle resolved X-ray photoelectron spectroscopy (ARXPS) studies

The adsorption of non-reactive alkanethiols and cyano-terminated thiols possessing the same chain length as well as their mixtures were examined on polycrystalline gold surfaces by angle resolved X-ray photoelectron spectroscopy (ARXPS). The adsorption behavior was investigated based on the chemical shift of the S 2p peak components. Besides the expected bound state of the thiols at the surface, non-bound molecules were detected with an increasing intensity at lower take-off angles. The layer composition does not entirely reflect the solution composition and the degree of adsorption is independent of the solution composition. A preferential adsorption of the methyl-terminated alkanethiol at the polycrystalline gold surface was measured. Received: 7 September 1998 / Revised: 16 February 1999 / Accepted: 21 February 1999     

Modification of indium tin oxide films by alkanethiol and fatty acid self-assembled monolayers: a comparative study

Indium tin oxide (ITO) substrates have been modified by alkanethiol and fatty acid self-assembled monolayers (SAMs). The SAMs were grown by dipping the cleaned surface into either a pure alkanethiol or a fatty acid dissolved in various solvents. They were characterized through contact angle, X-ray photoelectron (XPS) and infrared absorption-reflection spectroscopy (IRRAS). Their density and structural organization was found to greatly depend on the cleaning treatment of the ITO surface, the length of the alkyl chain, and, in the case of fatty acids, the concentration of the solution. XPS measurements brought evidence for the fact that, in the case of alkanethiols, the grafting mechanism was through the formation of ionic or covalent bonds involving thiolates. The most prominent result of this comparative study is that thiol-based SAMs are more strongly attached to the ITO substrate and better organized than fatty acids, which we attribute to the fact that the reaction of the ITO surface with fatty acids is more reversible than that with thiols.     

Diazonium salts: Stable monolayers on gold electrodes for sensing applications

The modification of gold electrodes with 4-carboxyphenyl diazonium salts to form stable layers for sensing applications is reported. Electrochemical reduction of 4-carboxyphenyl diazonium salts on gold electrodes yielded more stable layers than alkanethiol self-assembled monolayers in terms of extremes of electrode potential, sonication and with time. The application of the 4-carboxyphenyl modified electrodes for electrochemical sensing, which typically requires short chain alkanethiols on gold electrodes, is demonstrated via the covalent attachment of oligopeptides for the selective detection of Cu²⁺, Cd²⁺ and Pb²⁺. The diazonium salt/peptide modified gold electrodes not only had greater stability but also performed with lowest detected concentration to alkanethiol/peptide modified electrodes and with far greater sensitivity than the metal ion sensors when diazonium salt/peptide modified similar glassy carbon electrodes were employed.